This invention relates to flow cytometry and, more particularly, to flow cytometers for processing fluorescence emissions by lifetime-induced phase shifts.
Flow cytometry (FCM) is an important analytical tool for detecting characteristic fluorescence emission intensities from cellular components such as DNA, RNA, proteins, enzymes, and lipids stained with fluorescent dyes, antigenic determinants labeled with antibodies conjugated to fluorochrome, hybridized DNA sequences labeled with fluorescent probes, and the like, measured on a cell-by-cell basis at high speed. Fluorescence emission characteristics are detected, such as fluorescence signal intensity, signal width, and area, within specified wavelength regions defined by the fluorescence spectra as cells/particles intersect a cw laser or arc lamp excitation source. Conventional FCM has become an important clinical diagnostic and biomedical research tool, demanding ever-expanding capabilities to meet clinical and research needs. For example, the labeling of particles and cells with multiple fluorochromes is often required to provide a correlated analysis of cellular properties.
A major limitation of FCM is the availability of fluorescent dyes with common excitation regions, i.e., a single excitation source, with a resulting emission spectra that are sufficiently separated to permit measurement by multi-color detection methods. Examples of fluorochromes having overlapping emission spectra are shown in Table A. Fluorescence signal compensation using differential amplifiers, as described by Loken et al. 25 J. Histochem. and Cytochem. 899 (1977), has been employed to separate signals resulting from overlapping emission spectra in FCM applications, but with a loss in signal intensity depending upon the amount of "electronic compensation" needed to resolve the two signals.
TABLE A ______________________________________ LASER EXCITATION WAVELENGTH (nm) FLUORESCENT STAINS ______________________________________ UV Hoechst 33342, DAPI, DIPI, ANSA, DANSYL, NADPH, Ca.sup.++ Indicators, Coumarins 413 Brilliant Sulfaflavine, Fluorescamine, Mithramycin, Chromomycin 457 Mithramycin, Chromomycin, Acriflavine, FITC, FDA, Acridine Orange 488/514 Acriflavine, Acridine Orange, FITC, FDA, Rhodamine 123, Ethidium Bromide, Propidium Iodide, Phycoerythrin, Nile Red, SNARF-1 528/530 Phycoerythrin, Phyronin Y, TmRITC, Rhodamine B and 3G, Nile Red, Resorufin, SNARF-1 568 Texas Red, XRITC, Rhodamine 640, L. Rhodamine, Oxadicarbocyanin, Allophycocyanin ______________________________________
Another approach has used multiple excitation wavelengths to sequentially excite cells that are labeled with fluorochromes having separated excitation spectra that are sequentially detected on discrete photomultiplier tubes as described in Steinkamp et al., 62 Rev. Sci. Instrum. 2751-2764 (1991). This approach has increased the number of fluorescent dyes suitable for multilabeling experiments, but the instrument hardware has become increasingly complex.
Overlapping fluorescence spectra from batch solutions have been resolved using phase sensitive electronics to discriminate between emissions having different lifetimes, e.g., in spectrofluorometry, as originally described by Veselova, "Fluorometric Method for Individual Recording of Spectra in Systems Containing Two Types of Luminescent Centers," 29 Optics and Spectroscopy 617-618 (1970). Typical applications are described in McGown et al., "Phase-Resolved Fluorescence Spectroscopy," 56 Anal. Chem. No 13, pp. 1400A-1414A (November 1984) and Jameson et al., "The Measurement and Analysis of Heterogeneous Emissions by Multifrequency Phase and Modulation Fluorometry," 20(1) Appl. Spectrosc. Rev., pp. 55-103 (1984).
In accordance with the present invention, phase resolution techniques are applied to FCM to extend the capability of FCM to heterogeneous fluorescence emissions from at least two fluorochromes having overlapping emission (wavelength) spectra, but different fluorescence lifetimes. In addition to providing a new FCM measurement capability, i.e., the ability to resolve fluorochromes having overlapping emission spectra, it is recognized that phase-resolved measurements based on lifetime differences will improve measurement sensitivity, increase analytical resolution, and facilitate the interpretation of flow cytometric data by reducing background interferences from cellular autofluorescence, unbound (free) dye, nonspecific dye-binding, and Raman and Rayleigh scatter.
The ability to determine lifetime information from individual cell fluorescence emissions is also important since fluorescence decay/lifetime parameters provide additional information about fluorochrome/cell interactions. The fluorescence lifetime of dye molecules bound to specific components in cells and particles, such as DNA, can be influenced by local factors near the binding site, such as solvent polarity, energy transfer, excited-state reactions, and quenching. Thus, lifetime measurements add to flow cytometry the capability to probe structures on the molecular level in single cells, e.g. DNA structure.
Accordingly, it is an object of the present invention to use differences in fluorescence decay lifetimes to resolve signals from simultaneous fluorescence emissions having overlapping emission spectra arising from single-frequency excitation in a FCM.
It is another object of the present invention to enable an increased number of fluorochromes to be used for multilabeling experiments.
Yet another object of the present invention is to provide fluorescence decay lifetime as an output parameter from a FCM for use in characterizing fluorochrome/cell binding under a variety of conditions.
Still another object of the present invention is to improve the sensitivity and resolution of a FCM with regard to background interference fluorescence, such as intrinsic cellular autofluorescence, free/unbound dye, nonspecific staining, and Raman and Rayleigh scatter.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.